The objective of this project is to determine the potential for immunoactive foods in providing immunity to mucosal toxins or pathogens. Vaccine molecules expressed in food plants would provide an inexpensive and safe option for the vaccination of large populations. Moreover, availability of immunogens from renewable food sources (i.e., plants) would allow the safe production, convenient distribution and low technology maintenance of vaccines. This project focuses on a novel method of engineering and administering anti-toxic vaccines expressed in a food plant. A mouse model will be used to test the efficacy of a recombinant oral vaccine that will be expressed in transgenic alfalfa. The vaccine molecules will consist of the cholera toxin B chain or of chimeric constructs containing fragments of the cholera toxin B chain fused to an extracellular plant protein, cucumber acid chitinase (CAC). Transgenic alfalfa producing these antigens will be used to produce a standard mouse food. This food will be administered to mice, ad libitum, in short and long term studies to evaluate the development of local (gut-mucosal) and serum immune responses to cholera toxin antigens. The response of mice to orally ingested vaccine epitopes will be evaluated in the context of boosting immune responses to cholera toxin B chain and CAC epitopes following parenteral immunization as well as to priming immune responses to these antigens. Immune responses to the food borne vaccine will be compared to responses elicited by intestinal/gavage immunization and by parenteral immunization with CT and CAC. Gut mucosal secretions and sera will be collected at regular intervals to determine the nature of antibodies generated against cholera toxin and CAC epitopes. Anti-toxoid protective immunity will be assayed in ligated intestinal loops to test for protection against the watery diarrhea induced by whole cholera toxin. Comparison of immune responses to epitopes of the CAC protein resulting from feeding chimeric CAC or native CAC will be used to evaluate the oral adjuvant activity of cholera toxin peptides. Serum and mucosal antibodies will be tested for recognition of cholera toxin or CAC epitopes by Western blot and ELISA. Peptides derived from the cholera toxin B chain which do not induce hyporesponsive immune reactions in the gut may provide a means of stimulating mucosal immunity against other epitopes of recombinant food- borne vaccines. Such a result would effectively demonstrate multivalent oral immunogens in foods.